Carbon monoxide (CO) is a well know toxic gas, but emerging research also suggest potentially beneficial effects of temporarily increased levels of CO in attenuating deleterious effects of reactive oxygen species (ROS). To date the study and evaluation of the effects of CO have relied on detecting gross anatomical change of some observable parameter, such as infarct size in the studies of CO on ischemia/reperfusion or offline extracellular measurements using myoglobin as a colorimetric readout. These macroscopic and colorimetric outputs do not provide detailed information about cellular signaling pathways. Therefore we sought to develop a small molecule probe for the detection of CO. Similar to other gasotransmitters (NO and H2S) as well as other reactive small molecules such as H2O2, the use of small molecule turn-on fluorescent probes to investigate the pathways associated with these species has led to a greater understanding of these molecules.
Carbon monoxide (CO) is best known as a toxic gas inhaled from common sources such as smoke and car exhaust, but emerging studies show that this reactive small molecule is also continuously produced in the body from the breakdown of heme via the heme oxygenase enzymes (Tenhunen, R.; Marver, H. S.; Schmid, R. Proc. Nat. Acad. Sci. U.S.A. 1968, 61, 748-755; Maines, M. D. FASEB J. 1988, 2, 2557-2568; and Ryter, S. W.; Alam, J.; Choi, A. M. K. Physiol. Rev. 2006, 86, 583-650. Similar to the other major gasotransmitter molecules NO and H2S, CO is proposed to play significant roles in modulating responses to both chemical and physical stresses (Motterlini, R.; Otterbein, L. E. Nat. Rev. Drug Discovery 2010, 9, 728-743; Mann, B. E. Top. Organometallic Chem. 2010, 32, 247-285; Bilban, M.; Haschemi, A.; Wegiel, B.; Chin, B. Y.; Wagner, O.; Otterbein, L. E. J. Mol. Med. 2008, 86, 267-279; and Wu, L.; Wang, R. Pharm. Rev. 2005, 57, 585-630. In one example, exogenous and endogenous CO can provide protection against tissue damage during myocardial ischemia/reperfusion (Fujimoto, H.; Ohno, M.; Ayabe, S.; Kobayashi, H.; Ishizaka, N.; Kimura, H.; Yoshida, K.-i.; Nagai, R. Arterioscler. Thromb. Vasc. Biol. 2004, 24, 1848-1853), and to this end, CO releasing molecules (CORMs) based on transition-metal carbonyl complexes have been developed as potential therapeutics that allow for more targeted release of CO as compared to direct gas inhalation (Motterlini, R.; Otterbein, L. E. Nat. Rev. Drug Discovery 2010, 9, 728-743; Motterlini, R.; Clark, J. E.; Foresti, R.; Sarathchandra, P.; Mann, B. E.; Green, C. J. Circ. Res. 2002, 90, e17-e24; and Alberto, R.; Motterlini, R. Dalton Trans. 2007, 1651-1660).
Despite the important signal/stress dichotomy of CO, many aspects of its chemistry in biological systems remain elusive in part due to the lack of ways to selectively tracking this transient small molecule within intact, living biological specimens. Indeed, the primary methods for interrogating the biological effects of CO to date involve detecting a gross anatomical change of some observable parameter, such as infarct size in studying the effects of CO on ischemia/reperfusion, or offline extracellular measurements using myoglobin (Morimoto, Y.; Durante, W.; Lancaster, D. G.; Klattenhoff, J.; Tittel, F. K. Am. J. Physiol. 2001, 280, H483-H488; and Marks, G. S.; Vreman, H. J.; McLaughlin, B. E.; Brien, J. F.; Nakatsu, K. Antioxid. Redox Signaling 2002, 4, 271-277) or dirhodium-supported particles (Moragues, M. E.; Esteban, J.; Ros-L is, J. V.; Martinez-Manez, R.; Marcos, M. D.; Martinez, M.; Soto, J.; Sancenon, F. J. Am. Chem. Soc. 2011, 133, 15762-15772) for colorimetric readouts. We reasoned that the development of a CO-responsive small-molecule fluorescent probe would meet a critical need for new technologies to monitor this reactive small molecule in biological systems with spatial and temporal information. This approach has proved useful for studying the contributions of a variety of small signal/stress molecules in biological settings (Yang, Y.; Zhao, Q.; Feng, W.; Li, F. Chem. Rev. DOI: 10.1021/cr2004103; Kim, H. N.; Lee, M. H.; Kim, H. J.; Kim, J. S.; Yoon, J. Chem. Soc. Rev. 2008, 37, 1465-1472; Cho, D.-G.; Sessler, J. L. Chem. Soc. Rev. 2009, 38, 1647-1662; Jun, M. E.; Roy, B.; Ahn, K. H. Chem. Comm. 2011, 47, 7583-7601; and Du, J.; Hu, M.; Fan, J.; Peng, X. Chem. Soc. Rev. 2012, 41, 4511-4535), including NO (Kojima, H.; Nakatsubo, N.; Kikuchi, K.; Kawahara, S.; Kirino, Y.; Nagoshi, H.; Hirata, Y.; Nagano, T. Anal. Chem. 1998, 70, 2446-2453; Lim, M. H.; Xu, D.; Lippard, S. J. Nat. Chem. Biol. 2006, 2, 375-380; Yang, Y.; Seidlits, S. K.; Adams, M. M.; Lynch, V. M.; Schmidt, C. E.; Anslyn, E. V.; Shear, J. B. J. Am. Chem. Soc. 2010, 132, 13114-13116; and Kojima, H.; Hirotani, M.; Nakatsubo, N.; Kikuchi, K.; Urano, Y.; Higuchi, T.; Hirata, Y.; Nagano, T. Anal. Chem. 2001, 73, 1967-1973), H2S (Lippert, A. R.; New, E. J.; Chang, C. J. J. Am. Chem. Soc. 2011, 133, 10078-10080; Sasakura, K.; Hanaoka, K.; Shibuya, N.; Mikami, Y.; Kimura, Y.; Komatsu, T.; Ueno, T.; Terai, T.; Kimura, H.; Nagano, T. J. Am. Chem. Soc. 2011, 133, 18003-18005; Liu, C.; Pan, J.; Li, S.; Zhao, Y.; Wu, L. Y.; Berkman, C. E.; Whorton, A. R.; Xian, M. Angew. Chem. Int. Ed. 2011, 50, 10327-10329; Peng, H.; Cheng, Y.; Dai, C.; King, A. L.; Predmore, B. L.; Lefer, D. J.; Wang, B. Angew. Chem. Int. Ed. 2011, 50, 9672-9675; Qian, Y.; Karpus, J.; Kabil, O.; Zhang, S.-Y.; Zhu, H.-L.; Banerjee, R.; Zhao, J.; He, C. Nat. Commun. 2011, 2, 495; Yu, F.; Li, P.; Song, P.; Wang, B.; Zhao, J.; Han, K. Chem. Commun. 2012, 48, 2852-2854; and Montoya, L. A.; Pluth, M. D. Chem. Commun. 2012, 48, 4767-4769), and H2O2 (Lippert, A. R.; Van, d. B. G. C.; Chang, C. J. Acc. Chem. Res. 2011, 44, 793-804; Chang, M. C. Y.; Pralle, A.; Isacoff, E. Y.; Chang, C. J. J. Am. Chem. Soc. 2004, 126, 15392-15393; Miller, E. W.; Albers, A. E.; Pralle, A.; Isacoff, E. Y.; Chang, C. J. J. Am. Chem. Soc. 2005, 127, 16652-16659; Miller, E. W.; Tulyanthan, O.; Isacoff, E. Y.; Chang, C. J. Nat. Chem. Biol. 2007, 3, 263-267; Srikun, D.; Miller, E. W.; Domaille, D. W.; Chang, C. J. J. Am. Chem. Soc. 2008, 130, 4596-4597; Dickinson, B. C.; Chang, C. J. J. Am. Chem. Soc. 2008, 130, 9638-9639; Miller, E. W.; Dickinson, B. C.; Chang, C. J. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 15681-15686; Srikun, D.; Albers, A. E.; Nam, C. I.; Iavarone, A. T.; Chang, C. J. J. Am. Chem. Soc. 2010, 132, 4455-4465; Dickinson, B. C.; Peltier, J.; Stone, D.; Schaffer, D. V.; Chang, C. J. Nat. Chem. Biol. 2011, 7, 106-112; Abo, M.; Urano, Y.; Hanaoka, K.; Terai, T.; Komatsu, T.; Nagano, T. J. Am. Chem. Soc. 2011, 133, 10629-10637; and Song, D.; Lim, J. M.; Cho, S.; Park, S.-J.; Cho, J.; Kang, D.; Rhee, S. G.; You, Y.; Nam, W. Chem. Commun. 2012, 48, 5449-5451), but there are no reports of analogous indicators for CO. Herein, we present the design, synthesis, and biological evaluation of a new type of chemical reagent for selective CO detection in living cells by exploiting palladium-mediated carbonylation chemistry. Carbon Monoxide Probe 1 (COP-1) represents a unique first-generation chemical tool that features a robust turn-on response to CO with selectivity over reactive nitrogen, oxygen, and sulfur species and can be used to detect this gasotransmitter in aqueous buffer and in live-cell specimens.